Light Scattering Studies of the Effect of Ca2+on the Structure of Porcine Submaxillary Mucin 6. K. V A R M A , A. DEMERS, A. M. JAMIESON, J. BLACKWELL, A N D NEIL JENTOFT
Department of Macromolecular Science, Department of Biochemistry, Case Western Reserve University, Clevrland, Ohio 44106
SV NOPSIS
The effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCI,: these data are within experimental error of those for solutions in 0.1M NaC1. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z-average translational diffusion coefficient and the longest intramolecular relaxation time 7,.A plot of T~ vs (l/R,,)z-3 is linear, and consistent with plots of such data recorded for PSM in 0.1M NaCl and 6M GdnHCl solutions. However, the T~ values and the associated results for (Rhl)z-' in 0.5M CaCI, are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCI, solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.
I NTRODUCTlON
T h e activity of divalent cations in modifying the structure of mucin glycoproteins may be of great significance for the biological function in normal and diseased states.'-3 For example, it has been established th at calcium ions are present in high concentration inside the secretory granules of mucin-secreting cell^,^-^ and that calcium levels are elevated in the mucus of patients with cystic fibrosis.' Th e mechanism responsible for the characteristic abnormally thick mucus found in such individuals is not well understood. However, a cause and effect relationship between the control of the ionic environment on the mucosal surface and the viscoelastic properties of mucus is suggested by results indicating that mucus hydration is governed by a Donnan equilibrium process." This observation has prompted the idea that the ionic environment on the mucosal surface must play a &,
I Y Y O John W1le.y
B Sons, Inc.
CCC: O(H)6-.~5'L5/90/020441-08 $04.00 Biopolymers, Vol. 29, 441-448 (1990)
key role in the physiologic regulation of mucus hydration and hence on mucus rheology, i.e., the viscoelastic properties of mucus must be actively regulated not only by the neurohumoral mechanism th a t controls the release of mucin, but also by the movement of water and ions across the mucosa. Verdugo e t al.11~12 have suggested that a defect in the control of transepithelial hydroelectrolytic transport may be a common denominator in the pathophysiology of cystic fibrosis.6 Failure to remove calcium ions from the mucosal surface may lead to an abnormally thick, dehydrated mucus. Experimental results" indicate that an increase in extracellular Ca2 concentration can drastically decrease the diffusivity of the newly released mucin, resulting in extremely slow rates of swelling and a mucus gel that remains thick for long periods of time. Such findings have motivated a number of investigators to explore the potential role of Ca2&in modifying the conformation of mucin glycoproteins. Conflicting results on the effect of metal cation concentration on the structure of mucin glycoproteins have been reported. Chernick et al.'" +
441
442
VARMA ET AL.
proposed that Ca2+ ions act as cross-links between mucin molecules. However, Forstner et al.14 detected no evidence of precipitation, gelation, or polymerization of mucin glycoprotein in the presence of Ca2+.They observed a decrease in solution viscosity in the presence of 10-6-10-2M calcium chloride. From comparisons of their viscometric results, they have suggested that Ca2+ binds to negatively charged groups on goblet cell mucin (especially to the sialic acid carboxylates), and induces contraction or folding of these macromolecules. Marriot et al.15 have studied the effect of Ca2+ on canine tracheal mucin a t constant ionic strength of sodium chloride, and report a 47% increase in the elastic storage modulus. They attributed this effect to a conformational change in the mucin glycoprotein, since no change in the molecular charge density was observed. Several studies have led to the suggestion that Ca2+ ions form complexes with mucus. Deman et a1.16 proposed that Ca2+ forms ionic bridges between porcine gastric mucin molecules via the carboxylic acid groups of sialic acid moieties. However, other investigations on tracheal and cervical mucus glycoprotein indicate that these mucins are not dependent upon sialic acid for their rheological The existence of some sort of Ca2+-mucin complex has been proposed.20,21It has been noted further that Ca2+causes aggregation of a variety of normal glycoproteins, resulting in solutions of high turbidity, and produces abnormal patterns in electrophoresis.20*2' In the present paper we describe dynamic light scattering (DLS) investigations of fractionated samples of porcine submaxillary mucin (PSM) glycoprotein. Previous workers have employed DLS techniques to investigate the conformation and aggregation states of ~ u b m a x i l l a r y ,cervical,25 ~ ~ - ~ ~ and tracheal25 mucin in various solvents. The general consensus of these studies is that mucins are linear semiflexible chains of exceedingly high molecular weight constructed from subunit glycoproteins linked end to end by disulfide bonds. Because of the high density of oligosaccharide side chains, the peptide backbone is substantially expanded over that of nonglycosylated protein random coils. Lee et a1.26 reported the existence of two relaxation modes in mucin gels-one assigned to translational motion and the other to configurational dynamics. Steiner et al.27 have studied the effect of Ca2+ on canine tracheal mucin using DLS analysis under similar conditions to those used by Marriot et al.15 They observed a decrease in hydrodynamic radius with increasing concentration of calcium chloride,
which correlated with Ca2+-induced changes in mucin gel viscoelasticity. In the experiments reported below, we have investigated the influence of high concentrations of Ca2+ on mucin conformation and chain dynamics. These DLS studies were carried out using a 256-channel multi-tau correlator, which permits real-time analysis of the correlation function on four different sampling increments and facilitates high-resolution analysis of the distribution of DLS relaxation times. At small scattering vectors, qR, < 1, the DLS spectrum arises only from center-of-mass displacement, and these data are interpreted to give estimates of the average hydrodynamic radius R,, of the mucin. We have also extended our light scattering analyses to larger values of the scattering vector q such that qR, >> 1.0, where we are able2' to separate the relaxation spectrum into contributions arising from center-of-mass displacement and intramolecular (configurational) motions.
MATERIALS A N D METHODS Specimen Solution Preparation
Samples of porcine submaxillary mucin were purified as described by Shogren et al.24 and Varma et a1.28 The precipitated mucin product was dissolved in 0.15M NaCl and dialyzed against distilled water. After dialysis, solid guanidinium hydrochloride (GndHC1) was added to give a final concentration of 5M. Portions (100 mL) of this solution were chromatographed on a 9 x 80 cm column containing Sephacryl-1000, which was eluted with 5M GndHC1. The periodic acid shift assay of Mantle and Allen2' was used to estimate the carbohydrate content in the eluent. Fractions 57-80,81-100, 101-120, and 121-140 formed pools I, 11, 111, and IV, respectively, and contained 43.77, 39.29, 12.10, and 4.84% of the unfractionated specimen, respectively. The four pools were dialyzed against deionized H 2 0 for 72 h, and then concentrated using a Speedvac vacuum concentrator (Savant Instruments). Sodium azide (0.02%) was added to prevent bacterial growth. All liquid samples were stored under refrigeration. Light Scattering Analysis
Solutions of fractions I-IV of PSM glycoprotein were prepared containing different concentrations of calcium chloride. Each solution contained, in addition, 50 m M Tris/HCl buffer, pH 7 k 0.1, and
EFFECT OF Ca"
0.02% (w/v) sodium azide. The PSM solutions were filtered through 8.0-pm Millipore filters in order to remove dust. The buffered solvents had previously been clarified by filtration through 0.22-pm Millipore filters. Dilutions were made by adding known volumes of buffered solvents directly into the light scattering cell. The PSM solutions were also centrifuged for 1 h a t 6000 rpm immediately prior to light scattering analysis. All measurements were carried out a t 20 k 1°C using a photon correlator spectrometer (Brookhaven Instruments Corporation, Ronkonkoma, New York) with a Spectra Physics 10 mW He-Ne laser (632.8 nm) and a BI 2030AT 256-channel multi-tau correlator. The intensity autocorrelation function of the scattered light was measured for various combinations of concentration and scattering angles. The relaxation spectra were analyzed with the software package BI MSD ILT, which utilizes the multiexponential sampling approach of Ostrowsky et aLj30 to generate the distribution of relaxation constants G(I'). The accuracy of the multi-tau correlator and the reliability of the BI MSD ILT software have been verified by carrying out light scattering analysis of binary mixtures of anionic polystyrenes of differing molecular weights in the good solvent, toluene.
Data Analysis At scattering vectors qR 2 1.0, multiexponential sampling analysis of the distribution of relaxation times indicates two discrete relaxation mod^^.^^ The slower mode may be assigned to the translational diffusive motion of the polymers and has a mean decay rate rl given by
where G,( r) is the distribution of scattering amplitudes in the slow decay mode, and Bzis the z-average diffusion coefficient. The faster mode contains contributions from the intramolecular dynamics and has an average decay rate r,, which may be expressed as
where G,( r) is the distribution of scattering amplitudes in the fast decay mode and rcis the mean configurational relaxation time. In Eqs. (1) and (a), the scattering vector 4nii
4
=
e
sin x 2
(3)
ON T H E STRUCTURE OF PSM
443
where n is the solution refractive index, 0 is the scattering angle, and h = 632.8 nm is the wavelength of the incident laser radiation. The mean decay rate for the entire correlation function may then be evaluated as
where a , and a 2 are the fractional scattering amplitude of the slow and fast decays, respectively:
El
=
i*Gl(
r) d r and a, = ( a , + a,
=
iocr ) G2(
dT
i o c G ( r )d r = 1.0)
From the bimodal analysis, we evaluate the zaverage translational diffusion coefficient 0, from Eq. (1) and the mean intramolecular relaxation time T,, from
rc includes contributions from relaxation modes higher than rl. Extrapolation of rl to zero concentration yields @, from which we obtain the zaverage value of the inverse hydrodynamic radius (R using the Stokes-Einstein equation
')r
where 12, is the Boltzman constant, T is the absolute temperature, and qo is the solvent viscosity. By extrapolation to q R , z 1.0 and in the limit of infinite dilution, we may derive an estimate for r l : lim rc(qRh c+o
E
1.O)
= 7,
(7)
In practice, consistent with earlier we find that the concentration dependence of rc is negligible, and hence reported results for 7, are the mean of values obtained a t different concentrations. It is important to note here that the mucin fractions prepared for these studies are polydisperse samples of high molecular weight bipolymers. Thus static light scattering analysis requires careful determination of polymer concentrations to monitor solute losses during sample clarification procedure^.^^,^^ We find DLS analysis a more con-
444
VARMAETAL.
venient method to screen mucin samples for changes in molecular dimensions. A particular advantage is that the z-average value of the inverse hydrodynamic radius is less sensitive to polydispersity than the z-average radius of gyration.
RESULTS A N D DISCUSSION The weight average molecular weights for PSM (pool 11)dissolved in 0.25M CaC1, and 0.5M CaC1, were determined to be 12.5 x lo6 and 13.5 x lo6, respectively. These are within experimental error of the value of 12.4 X lo6obtained in our previous work for pool I1 dissolved in 0.1M NaC1,28where PSM appears to exist as some sort of dimeric aggregate.24,28We infer that, in dilute solution, Ca2+ ions do not lead to increased aggregation via ionic bridging. However, the hydrodynamic radius of PSM in CaC1, solution is significantly smaller than values estimated for the same sample in NaCl solution, suggesting a more compact chain conformation. DLS studies were therefore carried out to further examine these structural differences between PSM in the two solutions.
A typical electric field autocorrelation function is shown in Fig. l(a). These data were recorded a t 8 = 45" for PSM pool I in 0.033M CaC1, using multiple sampling times ( T ~= 30 ps, 7, = 60 p s , T~ = 120 ps, and T~ = 240 ps). Figure l(b) shows the corresponding distribution of relaxation frequencies [G ( r)]derived using the multiple exponential sampling technique. The G ( r ) data clearly show the existence of two discrete relaxation modes, i.e., G ( I?) = GI(I?) + G2(r). The computed mean relaxation frequencies are r = 215 r/s, = 105 r/s and = 420 r/s. Figure 2 shows the G(T) data derived from the scattering a t three additional angles [8 = 30" (A), 8 = 60" (B), and 8 = 90" (C)]. It can be seen that, as the scattering angle increases, the amplitude of the fast relaxation mode increases relative to that of the slow decay. Figure 3 demonstrates that r,/sin2 (6/2), which is proportional to the translational diffusive rate [cf. Eq. (3)], is independent of scattering angle. These data confirm that the contributions due to translational and internal modes have been successfully resolved a t all scattering angles. Extrapolation of r,/sin2( 8/2) to zero PSM concentration, as shown in Fig. 4, yields values for the limiting translational
r,
r,
7.9 A
Delay Time Increment (msec)
Figure 1. (a) Square of the electric field autocorrelation function of PSM I of porcine submaxillary mucin (1.645 X g/mL) dissolved in 3.3 X 10-2M CaCl,, 50 m M Tris/HCl buffer (pH 7.0). Multiple sampling time increments were used (7,= 30 p s , 72 = 60 ps, 73 = 120 ps, and 74 = 240 ps) a t 8 = 45 and 20°C. (b) Relaxation frequency distribution [G(T)] vs frequency (r)derived from Fig. l(a) [r, = 105 r/s, r2= 420 r/s, a,= 0.65, u2 = 0.351.
EFFECT OF Ca2' ON THE STRUCTURE OF PSM
445
I
fi 0
Y
(r)(rad/sec)
(T)(rad/sec)
g/mL) is 3.3 X 10-'M CaCl,, 50 Figure 2. Plots of G ( r ) vs r for PSM I (1.645 X mM Tris/HCl buffer (pH = 7.0) at three different scattering angles. (A) 6 = 30" (F, = 50 y/s, = 200 y/s, a, = 0.84, a, = 0.16). (B) 6 = 60" (F, = 195 y/s, = 970 y/s, a, = 0.67, a2 = 0.33). (C) 6 = 90" = 398 y / s , = 1843 y / s , a, = 0.64, a , = 0.36).
r2
(r,
r,
r2
--
4
1.0-
'V
1
I
al
s
--
tc)
'9
-C
1V
al 3 '
Q
v) Y
0.5-
N"
n
.-C
'0
?
X n
l k -
# 2.
OO
Y
RI
I
I
5
1 0
CONCENTRATIONx104(grnhnl)
.-C
c
Figure 4. A plot of r,/sin2(6/2) vs concentration of PSM I dissolved in 3.3 X 10-'M CaCl,, 50 m M Tris/HCl buffer (pH 7.0) at 20°C.
I .'
I
0.25
1
0.60
s in2(e/z) Figure 3. The translational diffusive decay rate r,/ sin2(8/2) (
Department of Macromolecular Science, Department of Biochemistry, Case Western Reserve University, Clevrland, Ohio 44106
SV NOPSIS
The effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCI,: these data are within experimental error of those for solutions in 0.1M NaC1. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z-average translational diffusion coefficient and the longest intramolecular relaxation time 7,.A plot of T~ vs (l/R,,)z-3 is linear, and consistent with plots of such data recorded for PSM in 0.1M NaCl and 6M GdnHCl solutions. However, the T~ values and the associated results for (Rhl)z-' in 0.5M CaCI, are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCI, solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.
I NTRODUCTlON
T h e activity of divalent cations in modifying the structure of mucin glycoproteins may be of great significance for the biological function in normal and diseased states.'-3 For example, it has been established th at calcium ions are present in high concentration inside the secretory granules of mucin-secreting cell^,^-^ and that calcium levels are elevated in the mucus of patients with cystic fibrosis.' Th e mechanism responsible for the characteristic abnormally thick mucus found in such individuals is not well understood. However, a cause and effect relationship between the control of the ionic environment on the mucosal surface and the viscoelastic properties of mucus is suggested by results indicating that mucus hydration is governed by a Donnan equilibrium process." This observation has prompted the idea that the ionic environment on the mucosal surface must play a &,
I Y Y O John W1le.y
B Sons, Inc.
CCC: O(H)6-.~5'L5/90/020441-08 $04.00 Biopolymers, Vol. 29, 441-448 (1990)
key role in the physiologic regulation of mucus hydration and hence on mucus rheology, i.e., the viscoelastic properties of mucus must be actively regulated not only by the neurohumoral mechanism th a t controls the release of mucin, but also by the movement of water and ions across the mucosa. Verdugo e t al.11~12 have suggested that a defect in the control of transepithelial hydroelectrolytic transport may be a common denominator in the pathophysiology of cystic fibrosis.6 Failure to remove calcium ions from the mucosal surface may lead to an abnormally thick, dehydrated mucus. Experimental results" indicate that an increase in extracellular Ca2 concentration can drastically decrease the diffusivity of the newly released mucin, resulting in extremely slow rates of swelling and a mucus gel that remains thick for long periods of time. Such findings have motivated a number of investigators to explore the potential role of Ca2&in modifying the conformation of mucin glycoproteins. Conflicting results on the effect of metal cation concentration on the structure of mucin glycoproteins have been reported. Chernick et al.'" +
441
442
VARMA ET AL.
proposed that Ca2+ ions act as cross-links between mucin molecules. However, Forstner et al.14 detected no evidence of precipitation, gelation, or polymerization of mucin glycoprotein in the presence of Ca2+.They observed a decrease in solution viscosity in the presence of 10-6-10-2M calcium chloride. From comparisons of their viscometric results, they have suggested that Ca2+ binds to negatively charged groups on goblet cell mucin (especially to the sialic acid carboxylates), and induces contraction or folding of these macromolecules. Marriot et al.15 have studied the effect of Ca2+ on canine tracheal mucin a t constant ionic strength of sodium chloride, and report a 47% increase in the elastic storage modulus. They attributed this effect to a conformational change in the mucin glycoprotein, since no change in the molecular charge density was observed. Several studies have led to the suggestion that Ca2+ ions form complexes with mucus. Deman et a1.16 proposed that Ca2+ forms ionic bridges between porcine gastric mucin molecules via the carboxylic acid groups of sialic acid moieties. However, other investigations on tracheal and cervical mucus glycoprotein indicate that these mucins are not dependent upon sialic acid for their rheological The existence of some sort of Ca2+-mucin complex has been proposed.20,21It has been noted further that Ca2+causes aggregation of a variety of normal glycoproteins, resulting in solutions of high turbidity, and produces abnormal patterns in electrophoresis.20*2' In the present paper we describe dynamic light scattering (DLS) investigations of fractionated samples of porcine submaxillary mucin (PSM) glycoprotein. Previous workers have employed DLS techniques to investigate the conformation and aggregation states of ~ u b m a x i l l a r y ,cervical,25 ~ ~ - ~ ~ and tracheal25 mucin in various solvents. The general consensus of these studies is that mucins are linear semiflexible chains of exceedingly high molecular weight constructed from subunit glycoproteins linked end to end by disulfide bonds. Because of the high density of oligosaccharide side chains, the peptide backbone is substantially expanded over that of nonglycosylated protein random coils. Lee et a1.26 reported the existence of two relaxation modes in mucin gels-one assigned to translational motion and the other to configurational dynamics. Steiner et al.27 have studied the effect of Ca2+ on canine tracheal mucin using DLS analysis under similar conditions to those used by Marriot et al.15 They observed a decrease in hydrodynamic radius with increasing concentration of calcium chloride,
which correlated with Ca2+-induced changes in mucin gel viscoelasticity. In the experiments reported below, we have investigated the influence of high concentrations of Ca2+ on mucin conformation and chain dynamics. These DLS studies were carried out using a 256-channel multi-tau correlator, which permits real-time analysis of the correlation function on four different sampling increments and facilitates high-resolution analysis of the distribution of DLS relaxation times. At small scattering vectors, qR, < 1, the DLS spectrum arises only from center-of-mass displacement, and these data are interpreted to give estimates of the average hydrodynamic radius R,, of the mucin. We have also extended our light scattering analyses to larger values of the scattering vector q such that qR, >> 1.0, where we are able2' to separate the relaxation spectrum into contributions arising from center-of-mass displacement and intramolecular (configurational) motions.
MATERIALS A N D METHODS Specimen Solution Preparation
Samples of porcine submaxillary mucin were purified as described by Shogren et al.24 and Varma et a1.28 The precipitated mucin product was dissolved in 0.15M NaCl and dialyzed against distilled water. After dialysis, solid guanidinium hydrochloride (GndHC1) was added to give a final concentration of 5M. Portions (100 mL) of this solution were chromatographed on a 9 x 80 cm column containing Sephacryl-1000, which was eluted with 5M GndHC1. The periodic acid shift assay of Mantle and Allen2' was used to estimate the carbohydrate content in the eluent. Fractions 57-80,81-100, 101-120, and 121-140 formed pools I, 11, 111, and IV, respectively, and contained 43.77, 39.29, 12.10, and 4.84% of the unfractionated specimen, respectively. The four pools were dialyzed against deionized H 2 0 for 72 h, and then concentrated using a Speedvac vacuum concentrator (Savant Instruments). Sodium azide (0.02%) was added to prevent bacterial growth. All liquid samples were stored under refrigeration. Light Scattering Analysis
Solutions of fractions I-IV of PSM glycoprotein were prepared containing different concentrations of calcium chloride. Each solution contained, in addition, 50 m M Tris/HCl buffer, pH 7 k 0.1, and
EFFECT OF Ca"
0.02% (w/v) sodium azide. The PSM solutions were filtered through 8.0-pm Millipore filters in order to remove dust. The buffered solvents had previously been clarified by filtration through 0.22-pm Millipore filters. Dilutions were made by adding known volumes of buffered solvents directly into the light scattering cell. The PSM solutions were also centrifuged for 1 h a t 6000 rpm immediately prior to light scattering analysis. All measurements were carried out a t 20 k 1°C using a photon correlator spectrometer (Brookhaven Instruments Corporation, Ronkonkoma, New York) with a Spectra Physics 10 mW He-Ne laser (632.8 nm) and a BI 2030AT 256-channel multi-tau correlator. The intensity autocorrelation function of the scattered light was measured for various combinations of concentration and scattering angles. The relaxation spectra were analyzed with the software package BI MSD ILT, which utilizes the multiexponential sampling approach of Ostrowsky et aLj30 to generate the distribution of relaxation constants G(I'). The accuracy of the multi-tau correlator and the reliability of the BI MSD ILT software have been verified by carrying out light scattering analysis of binary mixtures of anionic polystyrenes of differing molecular weights in the good solvent, toluene.
Data Analysis At scattering vectors qR 2 1.0, multiexponential sampling analysis of the distribution of relaxation times indicates two discrete relaxation mod^^.^^ The slower mode may be assigned to the translational diffusive motion of the polymers and has a mean decay rate rl given by
where G,( r) is the distribution of scattering amplitudes in the slow decay mode, and Bzis the z-average diffusion coefficient. The faster mode contains contributions from the intramolecular dynamics and has an average decay rate r,, which may be expressed as
where G,( r) is the distribution of scattering amplitudes in the fast decay mode and rcis the mean configurational relaxation time. In Eqs. (1) and (a), the scattering vector 4nii
4
=
e
sin x 2
(3)
ON T H E STRUCTURE OF PSM
443
where n is the solution refractive index, 0 is the scattering angle, and h = 632.8 nm is the wavelength of the incident laser radiation. The mean decay rate for the entire correlation function may then be evaluated as
where a , and a 2 are the fractional scattering amplitude of the slow and fast decays, respectively:
El
=
i*Gl(
r) d r and a, = ( a , + a,
=
iocr ) G2(
dT
i o c G ( r )d r = 1.0)
From the bimodal analysis, we evaluate the zaverage translational diffusion coefficient 0, from Eq. (1) and the mean intramolecular relaxation time T,, from
rc includes contributions from relaxation modes higher than rl. Extrapolation of rl to zero concentration yields @, from which we obtain the zaverage value of the inverse hydrodynamic radius (R using the Stokes-Einstein equation
')r
where 12, is the Boltzman constant, T is the absolute temperature, and qo is the solvent viscosity. By extrapolation to q R , z 1.0 and in the limit of infinite dilution, we may derive an estimate for r l : lim rc(qRh c+o
E
1.O)
= 7,
(7)
In practice, consistent with earlier we find that the concentration dependence of rc is negligible, and hence reported results for 7, are the mean of values obtained a t different concentrations. It is important to note here that the mucin fractions prepared for these studies are polydisperse samples of high molecular weight bipolymers. Thus static light scattering analysis requires careful determination of polymer concentrations to monitor solute losses during sample clarification procedure^.^^,^^ We find DLS analysis a more con-
444
VARMAETAL.
venient method to screen mucin samples for changes in molecular dimensions. A particular advantage is that the z-average value of the inverse hydrodynamic radius is less sensitive to polydispersity than the z-average radius of gyration.
RESULTS A N D DISCUSSION The weight average molecular weights for PSM (pool 11)dissolved in 0.25M CaC1, and 0.5M CaC1, were determined to be 12.5 x lo6 and 13.5 x lo6, respectively. These are within experimental error of the value of 12.4 X lo6obtained in our previous work for pool I1 dissolved in 0.1M NaC1,28where PSM appears to exist as some sort of dimeric aggregate.24,28We infer that, in dilute solution, Ca2+ ions do not lead to increased aggregation via ionic bridging. However, the hydrodynamic radius of PSM in CaC1, solution is significantly smaller than values estimated for the same sample in NaCl solution, suggesting a more compact chain conformation. DLS studies were therefore carried out to further examine these structural differences between PSM in the two solutions.
A typical electric field autocorrelation function is shown in Fig. l(a). These data were recorded a t 8 = 45" for PSM pool I in 0.033M CaC1, using multiple sampling times ( T ~= 30 ps, 7, = 60 p s , T~ = 120 ps, and T~ = 240 ps). Figure l(b) shows the corresponding distribution of relaxation frequencies [G ( r)]derived using the multiple exponential sampling technique. The G ( r ) data clearly show the existence of two discrete relaxation modes, i.e., G ( I?) = GI(I?) + G2(r). The computed mean relaxation frequencies are r = 215 r/s, = 105 r/s and = 420 r/s. Figure 2 shows the G(T) data derived from the scattering a t three additional angles [8 = 30" (A), 8 = 60" (B), and 8 = 90" (C)]. It can be seen that, as the scattering angle increases, the amplitude of the fast relaxation mode increases relative to that of the slow decay. Figure 3 demonstrates that r,/sin2 (6/2), which is proportional to the translational diffusive rate [cf. Eq. (3)], is independent of scattering angle. These data confirm that the contributions due to translational and internal modes have been successfully resolved a t all scattering angles. Extrapolation of r,/sin2( 8/2) to zero PSM concentration, as shown in Fig. 4, yields values for the limiting translational
r,
r,
7.9 A
Delay Time Increment (msec)
Figure 1. (a) Square of the electric field autocorrelation function of PSM I of porcine submaxillary mucin (1.645 X g/mL) dissolved in 3.3 X 10-2M CaCl,, 50 m M Tris/HCl buffer (pH 7.0). Multiple sampling time increments were used (7,= 30 p s , 72 = 60 ps, 73 = 120 ps, and 74 = 240 ps) a t 8 = 45 and 20°C. (b) Relaxation frequency distribution [G(T)] vs frequency (r)derived from Fig. l(a) [r, = 105 r/s, r2= 420 r/s, a,= 0.65, u2 = 0.351.
EFFECT OF Ca2' ON THE STRUCTURE OF PSM
445
I
fi 0
Y
(r)(rad/sec)
(T)(rad/sec)
g/mL) is 3.3 X 10-'M CaCl,, 50 Figure 2. Plots of G ( r ) vs r for PSM I (1.645 X mM Tris/HCl buffer (pH = 7.0) at three different scattering angles. (A) 6 = 30" (F, = 50 y/s, = 200 y/s, a, = 0.84, a, = 0.16). (B) 6 = 60" (F, = 195 y/s, = 970 y/s, a, = 0.67, a2 = 0.33). (C) 6 = 90" = 398 y / s , = 1843 y / s , a, = 0.64, a , = 0.36).
r2
(r,
r,
r2
--
4
1.0-
'V
1
I
al
s
--
tc)
'9
-C
1V
al 3 '
Q
v) Y
0.5-
N"
n
.-C
'0
?
X n
l k -
# 2.
OO
Y
RI
I
I
5
1 0
CONCENTRATIONx104(grnhnl)
.-C
c
Figure 4. A plot of r,/sin2(6/2) vs concentration of PSM I dissolved in 3.3 X 10-'M CaCl,, 50 m M Tris/HCl buffer (pH 7.0) at 20°C.
I .'
I
0.25
1
0.60
s in2(e/z) Figure 3. The translational diffusive decay rate r,/ sin2(8/2) (
